1. Field of the Invention
The present invention relates to a provirus DNA construct for producing retrovirus.
2. Description of the Background
The introduction of genes into cells is now used as an important research technique. For example, the function of a gene may be studied by introducing the same into cells. The gene may be introduced into primary culture cells or it may be introduced into reproductive cells to produce transgenic animals, mainly transgenic mice.
Alternatively, instead of studying the function of the gene, the function of a region encoding protein in the introduced gene may be studied. Also, the function of a gene region which regulates gene expression may also be studied.
In evaluating the progress of differentiation in a specific cell or the kinetics of a specified cell in vivo, a marker gene (for example, neomycin-resistant gene) is introduced into the specific cell to distinguish over other cells. This is also an important technique.
Next, it is likely that upon introducing a gene into productive cells to prepare a new transgenic animal, the transgenic animal may be utilized as livestock, an experimental animal or a pet animal.
Furthermore, it is also possible for humans to receive genetic treatment. Many genetic diseases caused by defects of a specific gene are known. It is also suspected that ordinary diseases such as cancer, autoimmune disease, diabetes, hypertension, infectious diseases, etc. are caused by deficiencies of a plurality of genes in combination. An attempt has thus been made to cure diseases by compensating for the defective gene or replacing the defective gene with a normal gene. At present, the introduction of a gene into productive cells of humans has been precluded due to moral considerations but attention has been focused, instead, on introducing genes into somatic cells for genetic treatment. The somatic cells to be so used are stem cells of the blood cell system, skin stem cells, hepatic stem cells, for example.
It is currently recognized that the most efficient method for introducing a gene into cells comprises using retrovirus. If circumstances require, a gene can be introduced into 100% of cells. In general, however, it is not easy to introduce a gene into primary culture cells or cells in vivo with a high efficiency as compared to the introduction into a cell line, even though retrovirus is used. In any case, it is important to contact a sufficient amount of retrovirus with cells, in order to enhance efficiency of gene introduction. Particularly in the case that the latter (primary culture cells or cells in vivo) is used, an amount of retrovirus greatly affects the efficiency.
When the cell line harbouring DNA for producing retrovirus particles is cultured, retrovirus can be obtained in the culture supernatant as virion. In such a state that retrovirus is suspended in medium, however, retrovirus is liable to be inactivated. Retrovirus is also liable to be inactivated by concentration operations. Accordingly, in order to efficiently perform gene introduction, it is necessary to obtain a medium having a high number of virion (which is referred to as titer and expressed by cfu/ml) having an activity of introducing a gene per unit volume. That is, virus-producing cells having a high titer are necessary.
Taking as an example gene introduction into blood cell stem cells used for genetic treatment using retrovirus, it is presumed that the stem cells into which a gene is to be introduced would be less than 0.1% of bone marrow cells, indicating that the density is very low. For this reason, a virus solution containing virion in an amount at least equivalent to, if possible, 10 times or 100 times 10.sup.9 to 10.sup.10 counts of bone marrow cells is generally required for bone marrow transplantation in a medium.
However, the upper limit of titer in the retrovirus-producing cell line for gene introduction obtained by introducing retrovirus vector pZipNeoSV(X)1 (C. L. Cepko et. al., Cell, 37, 1053, 1984) into helper cells .phi.2 (R. Mann et. al., Cell, 33, 153, 1983) ordinarily used is generally on the order of 10.sup.4 cfu/ml.
Provirus DNA encoding protein required for producing virion is incorporated into chromosomes of the retrovirus-producing cell line which is used for gene introduction, wherein components of virion are permanently produced by LTR (long terminal repeat) having the function of regulating DNA expression in the provirus. The components are produced from provirus DNA via mRNA; this mRNA per se is enveloped in these components to complete virion and virion is further released extracellularly. Natural virus infects other cells to release mRNA and enzymes in the virion. From this mRNA and enzymes, provirus DNA is formed and provirus DNA is further incorporated into chromosomes of the cells. By repeating this procedure, retrovirus proliferates.
However, retrovirus for gene introduction is defective in a part of the provirus DNA (packaging signal sequence, .phi.). Therefore, the virus components produced from provirus DNA fail to envelope mRNA likewise produced from provirus.
On the other hand, when a DNA construct wherein DNA other than LTR and packaging signal of provirus is removed and instead, a DNA construct having inserted therein a gene to be introduced (gene construct so designed as to incorporate an optional gene, retrovirus vector) is introduced into helper cells, mRNA formed from this DNA is selectively enveloped in the virus components formed from provirus DNA to produce virion; this virion infects other cells and DNA is transcripted from the enveloped mRNA and this DNA is incorporated into chromosome. Since DNA capable of producing virus is not present in the incorporated DNA, there is no chance to reproduce virion. By such procedures, an optional gene can be introduced into cells, using retrovirus.
As described above, a method for gene introduction using retrovirus may be considered to be logically ideal but involves various problems for practical use. Firstly, an amount of retrovirus produced by helper cells should be increased, as described above. Secondly, while it occurs with a poor frequency, provirus DNA or mRNA formed thereby and retrovirus vector DNA or mRNA formed thereby are recombined with each other, whereby provirus might sometimes take up packaging signal therein to form natural virus capable of persistent infection. Where the two have high homology on their sequence, this phenomenon occurs (R. A. Bosselman et. al., Mol. Cell. Biol., 7, 1797, 1987). Natural retrovirus has an ability of persistent infection so that there might be a danger of injuring the living body.
Thus, a need continues to exist for highly safe helper cells that do not allow natural virus to generate, and which overcome the above disadvantages.